A glycine zipper motif is required for the translocation of a T6SS toxic effector into target cells

Abstract Type VI secretion systems (T6SSs) can deliver diverse toxic effectors into eukaryotic and bacterial cells. Although much is known about the regulation and assembly of T6SS, the translocation mechanism of effectors into the periplasm and/or cytoplasm of target cells remains elusive. Here, we use the Agrobacterium tumefaciens DNase effector Tde1 to unravel the mechanism of translocation from attacker to prey. We demonstrate that Tde1 binds to its adaptor Tap1 through the N‐terminus, which harbors continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. Amino acid substitutions on G39xxxG43 motif do not affect Tde1–Tap1 interaction and secretion but abolish its membrane permeability and translocation of its fluorescent fusion protein into prey cells. The data suggest that G39xxxG43 governs the delivery of Tde1 into target cells by permeabilizing the cytoplasmic membrane. Considering the widespread presence of GxxxG motifs in bacterial effectors and pore‐forming toxins, we propose that glycine zipper‐mediated permeabilization is a conserved mechanism used by bacterial effectors for translocation across target cell membranes.


Introduction
In a complex microbial community, bacteria have evolved versatile secretion systems for the export or import of substrates across their membranes in response to different environmental cues. Each specialized protein secretion system (type I to X secretion system [TISS to TXSS]; reviewed in Costa et al, 2015;Christie, 2019;Palmer et al, 2020) can recognize specific substrates for secretion and translocation across one or multiple membranes. The type VI secretion system (T6SS) is a molecular weapon deployed by many Proteobacteria for pathogenesis, antagonism, or nutrient acquisition (Coulthurst, 2019). The T6SS effectors discovered so far exert functions in antibacterial, anti-eukaryotic, and metal acquisition (Russell et al, 2014;Hachani et al, 2016;Lien & Lai, 2017;Jurenas & Journet, 2021). The most established T6SS effectors are bacterial toxins, in which bacteria also produce cognate immunity proteins to prevent self-intoxication and toxicity in the sibling cells.
T6SS is a multiprotein complex, composed of at least 13 conserved core proteins resembling a phage tail structure, that extends from the cytoplasm to the outer membrane of the attacker cell (Cherrak et al, 2019;Wang et al, 2019). The T6SS machine consists of the Tss(J)LM membrane complex (MC), TssEFGK base plate (BP), TssBC contractile sheath, and Hcp-VgrG-PAAR puncturing device. The MC interacts with the BP (Durand et al, 2015;Cherrak et al, 2018), which serves as a docking site of VgrG-PAAR-effector complex to initiate the polymerization of the tail (Zoued et al, 2016). The tail is composed of the Hcp inner tube and TssBC outer sheath, whose biogenesis is regulated by TssA cap protein, and when triggered, the sheath contracts and ejects out the effector decorated puncturing device into extracellular milieu or target cells (Basler et al, 2012;Vettiger & Basler, 2016;Ali & Lai, 2022).
The T6SS has multiple strategies for delivering diverse effectors. On the basis of the known effectors and their transport mechanisms, effectors can be classified as "specialized" or "cargo" effectors (Cianfanelli et al, 2016;Cherrak et al, 2019). Specialized effectors are fused to either of the C-termini of three core structural proteins (Hcp, VgrG, or PAAR) while cargo effectors interact directly or require a specific chaperone/adaptor to be loaded into the lumen of the Hcp tube or onto the VgrG spike prior to secretion. Though diverse T6SS antibacterial effectors that act in the cytoplasm, membrane, or periplasm of the target cells have been reported (Russell et al, 2014;Lien & Lai, 2017;Jurenas & Journet, 2021), their mechanisms to breach outer and inner membranes for targeting cytoplasm of their targets still yet to be clarified.
A glycine zipper structure consisting of repetitive GxxxG motifs is commonly found in membrane-associated proteins (Kim et al, 2005) and bacterial toxins (Kim et al, 2004;Fonte et al, 2011). Glycine zipper motifs are known to be involved in the toxicity of some bacterial effectors for membrane channel formation. For example, the transmembrane domain (TMD) of a vacuolating toxin, VacA of Helicobacter pylori encodes three GxxxG motifs forming helix-helix packing interactions (Kim et al, 2004), which are required for the vacuolation and membrane channeling contributing to VacA toxicity (McClain et al, 2003). Type I secretion effectors CdzC and CdzD of Caulobacter crescentus and T6SS effector Tse4 of Pseudomonas aeruginosa, also possess glycine zipper motifs involved in the antibacterial activity (Garcia-Bayona et al, 2017;LaCourse et al, 2018). Expression of Tse4 disrupted the proton motive force of the inner membrane while CdzC and CdzD form surface aggregation for the contact-dependent killing of target cells. However, how glycine zipper motifs of Tse4 and CdzCD involved in toxicity remains unknown.
A T6SS-encoding locus is highly conserved in the genome of plant pathogenic bacterium Agrobacterium tumefaciens and the apparatus functions as an antibacterial weapon (Ma et al, 2014;Yu et al, 2020;Wu et al, 2021;Chou et al, 2022). We previously revealed that A. tumefaciens strain C58 deploys two Type VI DNase effectors (Tde1 and Tde2) as the major antibacterial weapons, in which the cognate immunity proteins (namely Tdi1 and Tdi2) prevent autointoxication (Ma et al, 2014). Both Tde1 and Tde2 harbor a C-terminal Novel toxin 15 (Ntox15) domain (Zhang et al, 2012) containing an HxxD catalytic motif required for its DNase activity (Ma et al, 2014). Tde1 requires its cognate chaperone/adaptor Tap1 for loading onto VgrG1 for secretion (Bondage et al, 2016).
By obtaining the uncoupling Tde1 variants that remain capable of binding to Tap1 for export but are deficient in membrane permeability, translocation, and interbacterial competition, we reveal the secretion and translocation mechanism of Tde1 from the attacker cell to the target cell. We show that the N-terminal region of Tde1 harboring repetitive glycine zipper motifs is sufficient for interacting with Tap1 for secretion. Once secreted, a conserved glycine zipper motif is necessary for translocation across target cell membranes. This finding demonstrates a new role of glycine zipper motif(s) in effector delivery into target cells.

Tde1 can cause DNase-independent growth inhibition in Escherichia coli
Our previous study showed that overexpression of Tde1 in A. tumefaciens C58 caused growth inhibition, and the immunity protein Tdi1 only partially protected against this cytotoxicity (Ma et al, 2014). We hypothesized that Tde1 has domains apart from the DNase domain that contributes to its toxicity. In addition to the C-terminal Ntox15 DNase domain (amino acid 99-247; Ma et al, 2014), Tde1 has a predicted transmembrane domain (TMD, 22-42; Fig 1A). Thus, three fragments of Tde1, that being the N-terminal, N-Tde1(1-97), and two C-terminal regions, C1-Tde1(50-278), and C2-Tde1(98-278) were tested for toxicity. To avoid confounding effects by the DNase activity, substitutions of catalytic residues (H190A, D193A) were introduced in the C1-Tde1 and the full-length wild-type (WT) Tde1 to become C1-Tde1(M) and Tde1(M), respectively ( Fig 1A). Ectopic expression in E. coli (DH10B) under an IPTG-inducible promoter showed that N-Tde1 was sufficient to inhibit growth ( Fig 1B). Tde1 (M), but not the C1-Tde1(M), is growth inhibitory. Although C2-Tde1 retains the wild-type DNase catalytic residues, it was not able to inhibit growth, suggesting the N-terminus is required for the DNase activity. Both C1-Tde1(M) and C2-Tde1 are expressed at levels similar to or higher than N-Tde1 or Tde1(M), indicating that their loss of growth inhibition is not due to the nonexpression of the proteins (Fig EV1A). This evidence suggests the N-terminal region of Tde1 is sufficient to confer toxicity under the conditions tested and that the C-terminal DNase domain requires the entire or part of the N-terminus for it to cause toxicity.
To test whether Tdi1, the immunity protein for the DNase toxicity of Tde1 (Ma et al, 2014), can also neutralize the N-Tde1 toxicity, the Tde1 variants were co-expressed with the Tdi1. The result shows that Tdi1 could not rescue the growth inhibition caused by the N-Tde1 and Tde1(M) (Figs 1C and EV1B). This indicates that Tdi1 cannot neutralize the N-terminus-mediated toxicity.
A glycine zipper motif in N-terminus of Tde1 is required for toxicity and enhanced membrane permeability To get an insight into the cause of growth inhibition by N-terminus of Tde1, we used N-Tde1 region as a query to search against the NCBI nonredundant (nr) database and identified Tde1 homologs encoded in the T6SS gene clusters of Agrobacterium/Rhizobium as well as tape measure proteins (TMP) encoded in genomes of Paraburkholderia/Burkholderia (Figs 1D and EV2A). We noticed the conservation of continuous copies of GxxxG motifs  in the Nterminus of Tde1, which resembles the glycine zipper motifs overrepresented in membrane proteins and reported to be involved in the membrane channel formation (Kim et al, 2005). Thus, we hypothesized that these repetitive glycine zipper motifs are involved in membrane permeability and N-Tde1 toxicity.
To verify the hypothesis, two highly conserved glycine residues at positions 39 and 43 of a glycine zipper motif were substituted with leucine (G39L and G43L), and the resulting N-Tde1 and Tde1 (M) variants were named as N-Tde1 GLGL and Tde1(M) GLGL , respectively. The growth analysis of E. coli DH10B cells by counting viable cells and OD 600 measurement showed that both N-Tde1 GLGL and Tde1(M) GLGL lost the ability to cause growth inhibition (Figs 2A and EV3A). Similar results were also observed when they were overexpressed in A. tumefaciens Δtde1 mutant (Fig EV3B), indicating that the G 39 xxxG 43 glycine zipper motif of Tde1 is required for the observed toxicity.
Next, we tested whether N-Tde1 can increase E. coli inner membrane permeability. To do so, we used the b-galactosidase activity assay to measure the entry of ortho nitrophenyl galactopyranoside (ONPG; 301 Da) into the cytosol. ONPG normally requires a functional permease LacY to enter into the cytosol but can enter if the inner membrane is permeabilized/compromised (Casteels et al, 1993;Epand et al, 2009). N-Tde1 and Tde1(M) as well as their glycine zipper substitution variants were expressed in E. coli BW25113ΔlacY (Baba et al, 2006) carrying b-galactosidase (pYTA-lacZ). The BW25113ΔlacY(pYTA-lacZ) complemented with lacY was used as a positive control. The E. coli cells were induced with IPTG to express Tde1 variants for 1 h and collected for ONPG uptake assay. This time point was chosen because there is no obvious difference in the number of viable cells among the strains tested (Figs 2A and EV3C). The results showed that cells expressing either N-Tde1 or Tde1(M) had similar b-galactosidase activity as LacYexpressing cells. By contrast, cells expressing N-Tde1 GLGL and Tde1 (M) GLGL only exhibited background-level activity as the negative controls ( Fig 2B). These results indicate that the N-Tde1 and Tde1 (M) are able to increase membrane permeability depending on the G 39 xxxG 43 motif. The data also suggest that the N-terminusmediated growth inhibition is caused by its ability to enhance inner membrane permeability through glycine zipper motifs.
To further analyze the extent of enhanced membrane permeabilization, cells from the same experiment were normalized to the same OD 600 and stained with Hoechst and propidium iodide (PI). Hoechst (616 Da) is a nucleic acid staining dye that is permeable to live Gram-negative bacterial cells while PI (668.4 Da) can only enter through a compromised inner membrane or dead cells. The PI/ Hoechst staining showed strong PI signals in cells expressing N-Tde1 and Tde1 (M) but no or few signals were detected in cells expressing N-Tde1 GLGL , Tde1(M) GLGL , or vector control, indicating that N-Tde1 is able to enhance membrane permeability to allow molecules with size 668.4 Da to pass ( Fig 2C). We next determined whether the N-terminus of Tde1 is bacteriostatic or bactericidal by growth recovery assay (Mariano et al, 2019). E. coli cells were induced with IPTG to express N-Tde1 or Tde1(M) and after 1 h, washed with fresh media without IPTG for continuous cultivation. We found that growth was recovered when cells were washed of the IPTG inducer, in contrast to the growth inhibition of cells with continuous IPTG induction ( Fig 2D). Collectively, the data suggest that the N-terminus of Tde1 is sufficient to facilitate membrane permeability for bacteriostatic toxicity, and such activity requires the conserved G 39 xxxG 43 glycine zipper motif.
The N-terminus of Tde1 is necessary and sufficient for Tap1 interaction Tap1 is the adaptor for loading Tde1 onto VgrG1 (Ma et al, 2014;Bondage et al, 2016). However, the region that Tde1 and Tap1 interact remains undefined. Thus, we performed a coimmunoprecipitation (co-IP) assay to identify the specific region of Tde1 that can interact with Tap1 in A. tumefaciens. The HA-tagged Tde1 variants were expressed in Δtde1 and anti-HA agarose bead was used to co-precipitate the interacting proteins followed by western blotting to detect Tde1 variants and Tap1. The results showed that the N-Tde1 and Tde1(M) interact with Tap1 but not the Cterminal variants, C1-Tde1(M) and C2-Tde1 ( Fig 3A). N-Tde1 GLGL and Tde1(M) GLGL remain capable of interacting with Tap1 ( Fig 3A). The results suggest that Tap1 interacts with Tde1 through the Nterminus and that the G39L and G43L substitution does not affect Tde1-Tap1 interaction.

The N-terminus of Tde1 is necessary and sufficient for secretion
Because N-Tde1 interacts with Tap1, we hypothesized that this region is required for Tde1 secretion. Thus, we performed a secretion assay by expressing the various HA-tagged Tde1 variants in Δtdei, a deletion mutant lacking both tde1-tdi1 and tde2-tdi2 toxin immunity pairs. Both cellular and extracellular fractions were collected to determine their expression and secretion, respectively. The results showed that all Tde1 variants containing N-terminus are secreted but not the C-terminus, C1-Tde1(M). The secretion is in a T6SS-dependent manner as secretion was essentially abrogated in ΔtdeiΔtssK, which lacks both tdei and tssk encoding the baseplate component. N-Tde1 GLGL and Tde1(M) GLGL are also stably expressed and secreted ( Fig 3B). The data suggest that N-terminus of Tde1 is necessary and sufficient for secretion and that the G39L and G43L substitution does not interfere with the secretion capacity of Tde1. Accordingly, Hcp secretion levels are highly correlated with Tap1-Tde1 interaction and secretion of Tde1 variants ( Fig 3B). The data also confirmed the requirement of the Tap1-Tde1 interaction for Tde1 secretion and supported our previous finding that Tde loading onto VgrG is critical for active T6SS secretion .
G 39 xxxG 43 motif of Tde1 is required for target cell delivery Because the G 39 xxxG 43 glycine zipper motif located in N-Tde1 increased the membrane permeability but was not required for secretion, we hypothesized that G 39 xxxG 43 is responsible for inserting Tde1 into the inner membrane and delivering it into the cytoplasm of target cells. Here, we engineered each of Tde1 variants fused to super-folder green fluorescence protein (sfGFP) with a flexible (GGGS) linker between Tde1 and sfGFP to avoid the Tde1 functional/structural interference by GFP. The sfGFP-fused Tde1 variants were expressed in A. tumefaciens Δtdei and ΔtdeiΔtssK mutants. The secretion assay results showed that both WT and G39L and G43L substitution of N-Tde1-sfGFP and Tde1(M)-sfGFP are secreted ( Fig EV4A). No or trace amounts of proteins were observed in the extracellular fractions of ΔtdeiΔtssk mutants, demonstrating that the secretion was T6SS dependent. C1-Tde1(M)-sfGFP protein signal could not be unambiguously determined in the cellular fraction due to the overlapping of its predicted protein band with cross-reacted proteins, and no corresponding C1-Tde1(M)-sfGFP band was detected in the extracellular fraction. The secretion assay of Tde1 variants fused with either HA or sfGFP concluded that N-Tde1 is necessary and sufficient for secretion and the G39L and G43L substitution does not affect Tde1 being secreted, which is correlated with the ability to interact with Tap1.
We next investigated the translocation of Tde1 variants by mixing A. tumefaciens Δtdei, expressing sfGFP-fused Tde1 variants, with E. coli cells expressing mCherry. A. tumefaciens expressing sfGFP only (Vector-sfGFP) was used as a negative control. After coculture, we imaged populations for mCherry (false colored in blue) and GFP (green) to detect E. coli and A. tumefaciens, respectively. We merged images to identify cyan-colored cells (overlayed blue and green signals), which represent E. coli cells with translocated ◀ Figure 1. Schematic domain organization, sequence alignment, growth inhibition assay of Tde1.

A
Schematic domain organization of Tde1 protein and its variants. The N-terminal repeated glycine zipper motifs (12-51) overlapping a predicted transmembrane domain (22-42) and Ntox15 DNase domain (99-247) are indicated. Tde1 and its variants with truncation or amino acid substitutions were illustrated. B, C (B) Growth inhibition assay of E. coli DH10B cells harboring pTrc200 vector or each of its derivatives expressing Tde1 variants with IPTG induction. (C) Growth inhibition assay of E. coli DH10B cells co-expressing the Tde1 variants expressed from pTrc200 plasmid and Tdi1 immunity gene expressed from pRL662 plasmid. Growth curve was determined at OD 600. Graphs of panels B and C show mean AE SD of three biological replicates (n = 3), each averaged with 3 technical repeats. One-way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison. Different letters indicate statistically different groups of strains (P value, 4.6 × 10 À5 and 5.19 × 10 À8 for panels B and C, respectively). D Multiple sequence alignment of N-Tde1 homologs were presented with highly conserved amino acid residues highlighted in yellow. The bacterial species, strain name, and locus number of Tde1 orthologs (Agrobacterium/Rhizobium) or tape measure proteins (Paraburkholderia/Burkholderia) are indicated on the left and right of aligned sequences. Two conserved glycine residues (G 39 , G 43 ) subjected to mutagenesis were indicated by the arrows above the sequences.
Source data are available online for this figure. Tde1 variants carrying sfGFP ( Fig 4A). We were able to observẽ 50% of cells with cyan fluorescence when A. tumefaciens expressing N-Tde1-sfGFP and Tde1(M)-sfGFP was co-cultured with E. coli mCherry whereas the GFP and mCherry signals were not overlapped in the E. coli cells co-cultured with A. tumefaciens strains expressing GFP only or sfGFP-fused C1-Tde1(M), N-Tde1 GLGL , Tde1(M) GLGL , respectively (Fig 4A and B). No cyan fluorescence was observed when N-Tde1-sfGFP and Tde1(M)-sfGFP were expressed in the ΔtdeiΔtssk mutant as the attacker (Fig EV4B).
The data suggest that Tde1 is translocated into target cells in a T6SS-and G 39 xxxG 43 -dependent manner. Because N-Tde1 GLGL -sfGFP and Tde1(M) GLGL -sfGFP could be secreted but not translocated into target cells, G 39 xxxG 43 motif is necessary for delivering Tde1 into the target cell.
G 39 xxxG 43 is critical for interbacterial competition but not for DNase activity To assess the role of the G 39 xxxG 43 motif for target cell intoxication in the context of interbacterial competition, A. tumefaciens C58 Δtdei expressing either Tde1-Tdi1, Tde1(M)-Tdi1, or single/ double G39L and G43L substitution variants, was competed with target E. coli (DH10B) cells. By counting the survival rate of E. coli prey cells, the data showed that A. tumefaciens Δtdei (Tde1-Tdi1) exhibits an antibacterial activity but not in the negative controls, the secretion deficient mutants ΔtssL and ΔtdeiΔtssK (Tde1-Tdi1; Fig 5A). No antibacterial activity could be observed for A. tumefaciens Δtdei expressing Tde1(M)-Tdi1, indicating the DNasemediated killing of E. coli. The antibacterial activity of Δtdei (Tde1 GLGL -Tdi1, Tde1 G39L -Tdi1, Tde1 G43L -Tdi1) was not detectable, similar to that of negative controls. We also performed interbacterial competition assays using A. tumefaciens strain 1D1609, which is susceptible to T6SS killing by C58 (Wu et al, 2019). The interbacterial competition between two A. tumefaciens strains was calculated by competitive index, which revealed the higher competitiveness of Δtdei (Tde1-Tdi1) and C58 against 1D1609 but no competitive advantage could be detectable for any of glycine zipper variants or Tde1(M) (Fig 5B). The observed antibacterial activity is T6SS-dependent because the killing activity of Tde1 was not observed when expressed in ΔtdeiΔtssK. The results indicate that G 39 xxxG 43 motif is required for interbacterial competition at both inter-or intra-species levels. We also performed a secretion assay of these A. tumefaciens attacker strains and all glycine zipper variants were secreted (Fig 5C). It is notable that Tde1 GLGL proteins accumulated at slightly lower levels while Tde1 G39L and Tde1 G43L had similar or even higher protein levels to that of Tde1 and Tde1 (M). Accordingly, Tde1 GLGL was secreted at lower levels.
To exclude the possibility that G39L and G43L substitution may influence its DNase activity, we performed in vivo plasmid DNA degradation assay. Tde1 and the variants were each expressed by the tightly controlled arabinose-inducible promoter for in vivo plasmid DNA degradation assay in E. coli BW25113 as described (Ma et al, 2014). It was observed that plasmid DNA was completely degraded in cells expressing Tde1, but not in the negative controls, the cells without arabinose induction nor cells expressing Tde1(M). Plasmid DNA was also degraded by Tde1 GLGL but not as complete as Tde1 while both Tde1 G39L and Tde1 G43L exhibit wild-type level DNase activity. (Fig 5D). The lower DNA degradation efficiency of Tde1 GLGL could be correlated with the barely detected Tde1 GLGL proteins (Fig 5D). We also found that the degree of plasmid DNA degradation is also correlated with the growth inhibition effect (Figs 5E and EV3D). The slight recovery of Tde1 GLGL from growth inhibition is consistent with the instability of Tde1 GLGL . The evidence that G39L and G43L substitutions abolished interbacterial competition but did not affect DNase activity and secretion of Tde1 suggest the G 39 xxxG 43 motif is necessary for delivering Tde1 across the inner membrane into the cytoplasm of target cells.

Discussion
Through the dissection of Tde1 DNase effector, we provide strong evidence for a role of the N-terminal glycine zipper motif(s) of Tde1 in delivering the T6SS effector into target cells. Here, we propose a model explaining the loading, firing, and translocation of Tde1 (Fig 6). In A. tumefaciens, Tde1 DNase activity is neutralized by Tdi1 by binding to C-terminal DNase domain while its N-terminal domain interacts with Tap1 for loading onto VgrG1 (Step 1). The VgrG1-Tap1-Tde1-Tdi1 complex is then recruited onto the membrane-associated baseplate, which serves as a docking site for polymerization of Hcp tube and TssBC sheath (Step 2). Upon TssBC sheath contraction (Step 3), Tap1 and Tdi1 may fall off and Hcp-VgrG-Tde1 puncturing device is then ejected for secretion. In contact with a target cell, Tde1 may be delivered to the periplasm of the target cell where Tde1 permeabilizes the inner membrane in a ◀ Figure 2. Growth inhibition and membrane permeabilization assays of glycine zipper mutants.

A
Growth inhibition assay of E. coli DH10B cells harboring pTrc200 vector or each of its derivatives expressing Tde1 variants with IPTG-inducible expression. The growth of E. coli was monitored by CFU counting every 1 h. B, C For membrane permeabilization assays, BW25113 WT alone or ΔlacY(pYTA-lacZ) cells harboring pTrc200 vector or each of its derivatives expressing Tde1 variants were carried out for (B) b-galactosidase activity assay to determine ONPG uptake, (C) propidium iodide permeability with cells treated with propidium iodide and Hoechst for detection by fluorescence microscope (Scale bar = 5 lm). For the quantification of cells with PI signals, a total of 6 randomly selected images obtained from two biological repeats were used to quantify the number of PI-stained cells/number of Hoechst-stained cells as indicated. D Bacteriostatic activity assay. E. coli DH10B cells harboring pTrc200 vector or each of its derivatives expressing Tde1 variants were cultured with or without IPTG induction for 1 h. The IPTG-induced cells were further centrifuged and resuspended in the fresh medium with or without IPTG. Cell density was measured again before continuous growth for additional 1 h.
Data information: Graphs of panels A, B, and D show mean AE SD of three biological replicates (n = 3), each averaged with 3 technical repeats. One-way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison. Different letters indicate statistically different groups of strains (P value, 1 × 10 À16 and 2 × 10 À16 for panels B and D, respectively). Source data are available online for this figure.
6 of 16 EMBO reports 24: e56849 | 2023 Ó 2023 The Authors A B Figure 3. The N-terminus of Tde1 is sufficient for interaction with Tap1 and secretion.
A Co-immunoprecipitation (Co-IP) in Agrobacterium tumefaciens. A. tumefaciens C58 Δtde1 harboring pTrc200 vector or its derivatives expressing HA-tagged Tde1 variants. Anti-HA resin was used to co-precipitate the Tde1 variants and Tap1. B Secretion assay for HA-tagged Tde1 variants. Western blot for the cellular and extracellular fractions of A. tumefaciens C58 Δtdei and ΔtdeiΔtssK expressing the HAtagged Tde1 variants. Hcp secretion was detected as a positive control for active T6SS secretion. Representative western blot results of three biological repeats were shown with antibody against HA, Hcp, or EF-Tu where EF-Tu serves as a loading and nonsecreted protein control. Protein markers are indicated in kDa.
Source data are available online for this figure. G 39 xxxG 43 motif-dependent manner (Step 5). Once delivered, Tde1 exerts its toxicity by attacking DNA for degradation (Step 6). T6SS cargo effectors often require the specific chaperone/adaptor for loading onto the puncturing device for secretion. Our previous findings demonstrated that Tap1, a DUF4123-containing protein, specifically interacts with Tde1 for loading onto VgrG1 for secretion (Ma et al, 2014;Bondage et al, 2016). We now show that the Nterminal region of Tde1 is necessary and sufficient for interaction with Tap1 for secretion and delivery into target cells. The evidence that Tde1 GLGL variant remains capable of binding to Tap1 for export but is deficient in membrane permeability and translocation demonstrates a distinct role of this G 39 xxxG 43 motif in target cell delivery. Among the 10 classes of the Ntox15-containing proteins, the majority of them including Tde1 belong to class I without detectable Nterminal domains (Fig EV2B). We identified the presence of glycine zipper motifs overlapping with the transmembrane domain (TMD) not only in N-terminal region of all Tde1 orthologs encoded in Rhizobiaceae but also in C-terminal region of tape measure proteins (TMP) encoded in genomes of Paraburkholderia/Burkholderia, likely as a prophage. TMP is a phage protein suggested to have a channel-forming activity (Roessner & Ihler, 1984, 1986) and as a determinant in connecting host inner membrane proteins for injecting phage genome into bacterial host cells (Cumby et al, 2015). Such conservation in Tde1 orthologs suggests that this glycine zipper-mediated delivery could be a common strategy deployed by these bacterial effectors for translocation across target cell membranes. It would be also interesting to investigate whether TMP also employs its C-terminal glycine zipper to mediate phage genome entry into host cells.
A role of N-terminal domain involved in the translocation of polymorphic toxins has been well documented in those contact-dependent growth inhibition (CDI) system and bacteriocins (Ruhe et al, 2020). However, little is known about the translocation mode of bacterial toxins delivered by other systems. Previous study in P. aeruginosa showed that VgrG-loaded Tse6-EagT6 complex is sufficient to translocate across a lipid bilayer in vitro (Quentin et al, 2018), suggesting a role of VgrG-effector complex itself in inserting across the inner membrane of target cells. A recent study further uncovered a widespread prePAAR motif in N-terminal TMDs of T6SS effectors involved in interaction with Eag family chaperone for export (Ahmad et al, 2020). The findings from the Tap1 and Eag chaperone-mediated T6SS toxins led us to propose that the bacterial toxins harboring a N-terminal TMD may be protected by its cognate chaperone/adaptor from insertion into membranes in the attacker cell. However, once the effector is injected into the periplasm of the target cell, specific motifs (such as glycine zippers or perhaps prePAAR) may insert into the inner membrane for the delivery into the cytoplasm. By an elegant in vitro translocation assay, a recent study discovered a N-terminal domain of a bacteriocin pyocin G is required for the import of its C-terminal nuclease toxin into the cytoplasm cross inner membrane (Atanaskovic et al, 2022). This inner membrane translocation domain (IMD) is distinct from the glycine zipper repeats identified in this study but also found conserved in other bacterial toxins including some of T6SS. Thus, a bacterial toxin directing its own translocation into target cells could be a general strategy used by bacteria for interbacterial competition.
A few membrane-permeabilizing T6SS toxins have been reported. The Vibrio cholerae VasX causes dissipation of membrane potential, leading to membrane permeabilization of target bacterial cells similar to the Tme effectors of V. parahaemolyticus, which represents a widespread family of T6SS effectors harboring C-terminal TMD for membrane disruption (Miyata et al, 2013;Fridman et al, 2020). On ◀ Figure 4. Translocation of Tde1 variants fused with sfGFP by Agrobacterium tumefaciens-Escherichia coli co-culture.
A Fluorescence microscopy for Tde1 translocation. A. tumefaciens C58 Δtdei expressing Tde1 variants fused with sfGFP (in green) and E. coli DH10B carrying mCherry (false colored in blue) were co-cultured for 20 h. A cyan fluorescence with merged blue and green signals represented the translocation of Tde1 variants from A. tumefaciens to E. coli (Scale bar = 5 lm). B The number of cells with overlayed GFP and mCherry fluorescence was quantified from a total of 6 randomly selected images obtained from three biological repeats (number of cells with cyan fluorescence/total E. coli cells counted).
Source data are available online for this figure.
▸ Figure 5. G 39 xxxG 43 glycine zipper motif of Tde1 is required for DNase-mediated killing of target cells during interbacterial competition.
A Interbacterial competition of Agrobacterium tumefaciens C58 Δtdei and ΔtdeiΔtssK expressing the Tde1 variants against E. coli cells was carried out on LB medium and E. coli survival rate was quantified by CFU counting. B Interbacterial competition between various A. tumefaciens C58 strains and A. tumefaciens 1D1609 on AK medium and the competition outcome was shown by competitive index. C Secretion assay for Tde1 and its variants co-expressed with its immunity protein Tdi1 in A. tumefaciens C58 Δtdei and ΔtdeiΔtssK. D In vivo plasmid DNA degradation assay. E. coli BW25113 carrying pJN105 empty vector or the derivatives expressing different variants of Tde1 was supplemented with 0.5% glucose ("À") or 0.2% L-arabinose ("+") for 3 h to either repress or induce Tde1 production. The plasmids were then extracted to observe the DNA degradation, and the bottom panel showed western blots of specific Tde1 protein bands. E Growth inhibition assay of Tde1 and its variants. E. coli BW25113 cells were induced by adding 0.2% L-arabinose for Tde1 production. The OD 600 values were measured every 15 min. The OD 600 values of the 4 h post-L-arabinose induction were analyzed for statistical analysis. Graphs show mean AE SD of three biological repeats.
Data information: Western blots were detected with a specific antibody against Tde1, Hcp, or EF-Tu serving as a loading and nonsecreted protein control. Protein markers are indicated in kDa. Data in panel A are mean AE SD of four biological repeats of two independent experiments (n = 4). Panels B and E show mean AE SD of three biological repeats (n = 3). One-way ANOVA was used for the analysis of statistical significance followed by the Fisher's least significant difference (LSD) test for panels A and B while the Tukey's test was done for panel E. Different letters indicate statistically different groups of strains (P value, 3.63 × 10 À4 , 2.70 × 10 À3 , 2.3 × 10 À15 for panels A, B, and E, respectively). Results in panels C and D are representative of three biological repeats. Source data are available online for this figure. the other hand, Tse4 disrupts the membrane potential and forms a cation-selective pore without membrane permeabilization where the pore cannot even allow the permeability of a relatively smaller molecular weight (ONPG, 300 Da;LaCourse et al, 2018). Distinct from these toxins in which they confer pore-forming activity for toxicity, the role of glycine zipper(s) of Tde1 appears to enhance membrane permeability for bringing the toxin domain into target cell cytoplasm because Tde1(M) with complete glycine zipper motifs but the loss of DNase activity did not show interbacterial competition activity against E. coli or A. tumefaciens under conditions tested (Fig 5A and B; Ma et al, 2014).
To date, no structural information is available for Ntox15 superfamily proteins where Tde1 belongs. While N-terminus of Tde1 lacks sequence similarity to any of those known pore-forming toxins, structural similarity to pyocin S5 and colicin Ia could be predicted by Phyre2 (Fig EV5A). Further structural modeling showed the structural similarity of two helixes containing consecutive glycine zipper motifs of N-Tde1 (10-62) to the pore-forming domain of pyocin S5 (Behrens et al, 2020;Fig EV5B-D). Pyocin S5 can cause ATP leakage and PI permeability (Ling et al, 2010) potentially to the inner membrane after translocation through FptA and TonB1 (Behrens et al, 2020). Tde1 allows the passage of a relatively larger molecule, PI (668 Da), suggesting that its N-terminal glycine zipper(s) may form larger pores similar to pyocin S5. G 39 xxxG 43 motif plays no role in DNase activity of Tde1 but is crucial for its protein stability. Tde1 with the substitution of one of Figure 6. Proposed model of the loading, firing, and translocation of Tde1. The Tde1 translocation is proposed through six steps.
Step 1: Tde1 forms a complex with Tdi1 and Tap1 in the attacker cell.
Step 3: Hcp tube and TssB/C sheath polymerize on the Tde1-loaded VgrG/baseplate while Tdi1 and Tap1 fall off with unknown mechanisms before or upon firing.
Step 4: TssBC sheath contracts and ejects Tde1 into the target cell periplasm or cytoplasm.
Step 5: The glycine zipper(s) on the N-terminus of Tde1 permeabilize the target cell membrane.
Step 6: Intact or truncated Tde1 proteins attack DNA for degradation in the target cell.

Ó 2023 The Authors
EMBO reports 24: e56849 | 2023 the two glycine residues to leucine retains the stability of intact proteins, but Tde1 is prone to truncations and degradation when both glycine residues are substituted to leucine. The instability is most evident when ectopically expressed in E. coli and when retaining DNase activity (Figs 5 and EV1A). Single glycine substitution (Tde1 G39L and Tde1 G43L variants) does not influence protein stability may suggest that the adjacent glycine residues (G 35 or G 47 ) are sufficient to compensate the loss of one glycine of G 39 xxxG 43 motif for structural integrity in both variants. The importance of G 39 xxxG 43 motif in Tde1 protein stability is consistent with the role of glycine zippers in structural impact (Kim et al, 2005). However, the integrity of G 39 xxxG 43 motif is critical for interbacterial competition because both Tde1 G39L and Tde1 G43L variants do not exhibit detectable antibacterial activity to either E. coli or A. tumefaciens (Fig 5A and B). These results suggest the role of G 39 xxxG 43 motif in delivering Tde1 across the inner membrane into the cytoplasm of target cells.
It is striking to observe such a high percentage of cells (~50%) representing N-Tde1-sfGFP and Tde1(M)-sfGFP translocation from A. tumefaciens into E. coli cells expressing mCherry (Fig 4). Adding the flexible GGGS linker between sfGFP and Tde1 that retain both Tde1 secretion activity and GFP fluorescence may be the key to the success of this translocation experiment. Interestingly, we also observed many GFP foci from A. tumefaciens cells expressing translocation-competent N-Tde1-sfGFP or Tde1(M)-sfGFP while others including E. coli cells with GFP signals were found to be uniformly distributed throughout the cells. Based on the role of glycine zippers in interacting with membrane, we propose that Tde1 proteins may preferentially bind to the microdomain of the cytoplasmic membrane, which was recently found in A. tumefaciens (Czolkoss et al, 2021). We also found that Tde1 proteins (either tagged with HA or GFP, Figs 3, EV1, and EV4A) are prone to truncation especially when they are ectopically expressed in E. coli or when Tdi1 is absent or not equivalent. Thus, it is possible that Tde1-GFP proteins are truncated after translocation into E. coli cells, in which most GFP signals are emitted from free GFP instead of Tde1-GFP. The stability of free GFP derived from translocated Tde1-GFP may also explain the high percentage of E. coli cells exhibiting overlayed GFP/mCherry signals. There is evidence that the truncation of T6SS effectors is critical for toxicity (Pei et al, 2020). Future work to investigate how Tde1 interacts with membrane and dissects the region required for DNase activity shall shed light to understand the biological significance and mechanisms underlying this interesting observation.
With the knowledge of effector translocation mechanisms, the bacterial protein secretion apparatus also offers a strategy for delivering heterologous proteins to specific cells. T6SS is a promising vehicle for such purpose because effectors or secreted proteins appear to be delivered with their folded or partially folded form, unlike those to be transported as unfolded forms in most of the other specialized secretion systems (Costa et al, 2015). Engineering T6SS carriers such as VgrG spikes to carry exogenous effector proteins into target cells are feasible but challenging (Ho et al, 2017;. By using a truncated variant of PAAR, a recent study showed delivering exogenous T6SS effectors and Cre recombinase for genetic modification in the target cells (Hersch et al, 2021). Our success in using N-Tde1 in the delivery of sfGFP proteins into target E. coli cells where they exert fluorescence also suggests potential applications of N-Tde1 for the delivery of proteins of interest such as genetic modifiers. This strategy provides advantages over transforming foreign DNA for expressing a protein of interest from creating undesired genome manipulation.

Strains and growth conditions
The strains and plasmids used in this study are listed in Appendix Tables S1 and S2. The E. coli strains used in this study are BW25113 and DH10B. All the A. tumefaciens strains were cultured on 523 medium (Kado & Heskett, 1970) at 28°C unless stated. The E. coli strains were cultured on Luria Bertani (LB) medium (10 g L À1 NaCl, 10 g L À1 tryptone, and 5 g L À1 yeast extract) at 37°C unless stated. Where appropriate, the media were supplemented with 100 lg ml À1 spectinomycin (Sp), gentamycin (Gm) 25 lg ml À1 (for E. coli) and 50 lg ml À1 (for A. tumefaciens), 50 lg ml À1 ampicillin (Amp), 50 lg ml À1 kanamycin (Km), 1 mM Isopropyl b-d-1thiogalactopyranoside (IPTG).

Growth inhibition assay
For growth inhibition assay of IPTG-inducible expression of Tde1 and its variants, E. coli (DH10B) harboring pTrc200 vector or the derivatives expressing Tde1 variants were grown overnight in LB medium supplemented with Sp prior to 1:30 dilution in a fresh medium and incubated for 2 h at 37°C with 250 rpm. After 2 h, the cultures were normalized to OD 600 0.1 in LB with 1 mM IPTG for continuous culture in the same growth condition. The growth of E. coli was monitored for OD 600 every 1 h using ULTROSPECâ 10 cell density meter (Biochrom, UK) or viable cell by counting colony forming units (CFUs) on LB agar containing Sp. For growth inhibition assay of arabinose-inducible expression, E. coli BW25113 harboring pJN105 vector or the derivatives expressing Tde1 variants were used. Overnight cultures of E. coli cells were adjusted to an OD 600 of 0.1 in 200 ll LB with 0.2% L-arabinose in a 96-well plate. The OD 600 values were measured by the Synergy H1 microplate reader (Agilent Technologies, USA) with agitation at 37°C. The OD 600 values or CFUs of indicated time points were used to calculate mean AE SD of three biological repeats. One-way analysis of variance (ANOVA) was used for the analysis of statistical significance followed by the Tukey's multiple comparison.

In vivo plasmid DNA degradation assay
The in vivo plasmid DNA degradation assay was performed as described (Ma et al, 2014) with minor modifications. Briefly, overnight cultures of E. coli BW25113 carrying pJN105 vector or the derivatives expressing Tde1 variants were adjusted to an OD 600 of 0.3 in 4 ml LB with 0.5% D-glucose or 0.2% L-arabinose. After induction for 3 h, bacterial cells normalized by OD 600 were collected for plasmid DNA extraction and western blot analysis. The plasmids were then extracted and applied to 0.6% agarose gel electrophoresis to detect DNA degradation. The OD 600 values were measured by DEN-600 photometer (Biosan, Latvia) every hour.
b-Galactosidase and viability assays for ONPG update b-galactosidase assay was performed as described (Saint Jean et al, 2018) with minor modifications. BW25113 wild-type, BW25113ΔlacY(pYTA-lacZ), or BW25113ΔlacY harboring pTrc200 vector or the derivatives expressing Tde1 variants were grown overnight and refreshed to a fresh medium as stated for growth inhibition assay. After subculture for 2 h, the cells were induced with 1 mM IPTG, and incubated for one more hr. Part of the culture was adjusted to OD 600 = 0.3 in Z-buffer and the Intracellular bgalactosidase activity was measured by mixing 100 ll of 4 mg ml À1 ONPG with 900 ll of the cells and incubation at room temperature for 10 min prior to measurement at OD 420 . The remaining cells were normalized to OD 600 0.3 in 0.9% sterile saline and after serial dilution, 10 ll was spotted on the LB plate without antibiotics to recover the viable cells. Data from OD 420 were used to calculate mean AE SD of three independent experiments. One-way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison.

Co-immunoprecipitation (Co-IP)
The co-IP was performed according to the manufacturer's recommendations of EZview red Anti-HA agarose (Sigma-E6779) with minor modifications. To identify Tap1-interacting domain of Tde1, the HA-tagged Tde1 variants were expressed from pTrc200 plasmid. For co-IP in A. tumefaciens, C58 Δtde1 cells expressing the Tde1 variants grown in 523 medium overnight were resuspended in a 1:30 ratio to a fresh medium and incubated at 25°C for 3 h followed by 1 mM IPTG induction for additional 3 h. After 6 h postincubation, the cells were normalized to OD 600 of 5 per ml in ice-cold PBS buffer (pH 7.4). After cell lysis by lysozyme treatment and sonication, the lysate was centrifuged and a 100 ll aliquot of the lysate was saved for the input fraction. The remaining 900 ll lysate was mixed with 25 ll of pre-equilibrated Ezview red Anti-HA agarose and incubated at 4°C for 1 h. The beads were then washed 3 times with ice-cold PBS buffer and the proteins bound to the beads were eluted with 100 ll of 2× SDS sample loading buffer. Similarly, the aliquoted input fraction was mixed with an equal volume of 2× SDS sample loading buffer for analysis by western blotting.

Secretion assay
Type VI secretion assay was performed in 523 medium as described previously . Briefly, A. tumefaciens strain was cultured overnight in 523 medium and normalized to OD 600 of 0.2 in a fresh medium. After 6 h of culturing, the secreted proteins were collected by centrifuging at 10,000 g for 5 min. The resulting pellet was adjusted to OD 600 of 10 as a cellular fraction. The culture supernatant was filtered with 0.22 lm Millipore filter membrane, and the resulting filtrate was subjected to TCA precipitation (Wu et al, 2008) and referred to as an extracellular fraction.

Interbacterial competition assays
For interbacterial competition with E. coli (target), A. tumefaciens strain (attacker) was grown overnight at 28°C in 523 broth with appropriate antibiotics if needed. E. coli DH10B harboring pRL662 plasmid was grown at 37°C in LB broth with Gm. After harvesting and washing the cells in 0.9% saline, the attacker to target cell density was adjusted to 30:1 (OD 600 = 3: 0.1) and the mix was spotted on LB medium containing 1.5% (wt/vol) agar. After incubation of the mixed strains for 16 h at 28°C, the spot was resuspended in 0.9% saline, serial diluted, and spotted on a gentamycin-containing LB agar square plate at 37°C to calculate E. coli survival rate by CFU counts. Similar procedure was used when using A. tumefaciens strain 1D1609 as a target, which was grown at 28°C in 523 broth prior to competition. The competition was carried out on AK medium for 16 h at 28°C with CFU counting at both initial and final time points by the selection of C58 strains with Sp resistance and 1D1609 with Gm resistance. To calculate the competitive index, CFUs of A. tumefaciens attacker C58 strain were divided by the CFUs of target 1D1609 strain at both 0 h and 16 h, and the ratio of C58/1D1609 at 16 h was divided by the ratio of C58/1D1609 at 0 h to obtain competitive index. One-way ANOVA was used for the analysis of statistical significance followed by the Fisher's least significant difference (LSD) test.

Fluorescence microscopy
For propidium iodide and Hoechst staining, E. coli cells (BW25113) harboring pTrc200 vector or derivatives expressing Tde1 variants were grown overnight and refreshed to a fresh medium as stated for growth inhibition assay. After subculture for 2 h, the cells were induced with 1 mM IPTG for 1 h and OD 600 equivalent to 0.3 was collected in 1 ml PBS and stained with Hoechst 33342 (H3570) to a final concentration of 12.3 and 1 lg ml À1 propidium iodide (2208511) and incubated for 2 min in dark. Finally, 2 ll was spotted on 2.5% agarose pad.
For the translocation experiment, the sfGFP-fused Tde1 variants were expressed in A. tumefaciens Δtdei cells (attacker). E. coli (target) cells were labeled with mCherry (false color blue) expressed from pBBRMCS2. A. tumefaciens attacker cells were cultured in 523 broth overnight, and E. coli target cells were separately cultured on LB broth. Overnight cultured attacker and target cells were mixed at a 5:1 ratio (OD 600 = 1.0:0.2), and 10 ll of the mix was cultured on an LB agar plate without IPTG. After 20 h of co-culture, the cells were washed with 100 ll PBS and 2 ll of suspension was spotted on the 2.5% agarose pad on a microscopic slide. The translocation signal was detected as the merge of GFP and mCherry (false colored in blue), which is of cyan fluorescence.
Fluorescence microscopy was performed using Axio Observer 7 (Zeiss, Germany) microscope equipped with an Axiochem 702 digital camera and a Plan-Apochromat 100×/1.4 Oil DIC H objective. Exposure times were adjusted to 20 ms for Phase, 50 ms for Hoechst, 150 ms for PI, 200 ms for GFP, and 5,000 ms for mCherry. Multiple images were taken from different fields and all the experiments were performed at least in triplicate and a representative image is shown. Images were analyzed by using ZEN 2.3 (blue edition) software.

Domain prediction and analysis
Full-length Tde1 (1-278) was used as a query for conserved domain search on the conserved domains database (CDD; Lu et al, 2020) of the National Center for Biotechnology Information (NCBI). Prediction of the transmembrane domain was done using the PRED-TMR2 (Pasquier et al, 1999). The Tde1 homologs and tape measure proteins (TMPs) for the multiple sequence alignment were obtained by BLAST search of N-Tde1 (1-97) against the NCBI nonredundant database (nr) with representative sequences selected for multiple sequence alignment. The domain architectures of the Ntox15 domain-containing proteins were obtained using the full-length Tde1 against the Conserved Domain Architectural retrieval tool (CDART) of NCBI. The information of gene clusters encoding Tde1 homologs and TMPs including upstream and downstream three genes was retrieved from their respective genomes. N-Tde1(1-97) was used as a query for structural prediction on a Phyre2 (Kelley et al, 2015). Three-dimensional structure modeling was done using Phyre2 in intensive modeling mode. Crystal structure served as the best template for the N-terminus, and the percentage of confidence for three-dimensional structure modeling is indicated in the legends of corresponding figures. The structural graphics were generated by using ChimeraX 1.1 (Goddard et al, 2018).

Data availability
No large primary datasets have been generated and deposited.
Expanded View for this article is available online.